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Circadian Rhythm of Free Melatonin in Human Plasma¹

Department of Obstetrics and Gynecology, University of Adelaide Medical School, Adelaide, South Australia 5005, Australia

Address all correspondence and requests for reprints to: Dr. D. J. Kennaway, Department of Obstetrics and Gynecology, University of Adelaide Medical School, Adelaide, South Australia 5005, Australia.

	Abstract

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The appearance of melatonin in saliva in concentrations up to 70% lower than those in blood has led to the suggestion that melatonin is bound to plasma protein and that saliva levels reflect the circulating free hormone. To test this directly, melatonin was measured in human plasma from 10 subjects after ultrafiltration through Centrifree micropartition tubes and compared to saliva melatonin levels in samples collected simultaneously. Melatonin was detected in the protein-free fraction and increased throughout the night in parallel with the saliva melatonin level. Peak concentrations ranged from 45–200 pmol/L (mean ± SEM, 106 ± 17 pmol/L) and averaged 23% of the total melatonin level. Across all samples, the correlation between the saliva levels and the free hormone levels was significant (r = 0.84; P < 0.05). These results provide the first direct evidence that endogenous melatonin is bound to plasma proteins and that saliva melatonin generally reflects the levels of this binding.

	Introduction

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THE RHYTHMIC production of melatonin by the pineal gland and its secretion into the blood were described in humans over 20 yr ago (1). Since that time, there have been hundreds of studies incorporating the measurement of melatonin in blood using bioassays, mass spectrometry, and RIA. A feature of all of these assays is the fact that they have measured the total amount of melatonin in samples and have not addressed the potential importance of alterations in binding of the hormone to plasma proteins.

It is well established for a large number of hormones that protein binding has the potential to modulate the amount of hormone that is presented to target cells. There has been only one comprehensive study of plasma protein binding of melatonin; these researchers studied the binding of tritiated melatonin to plasma and concluded that melatonin was bound to human albumin (2). Indirect evidence that has been assumed to confirm this result has come from measurement of melatonin in human saliva (3); as this fluid is devoid of albumin and globulins, the saliva/plasma ratio should reflect the free/bound ratio in blood. Despite the availability of sensitive melatonin assays for many years, however, this relationship has never been tested experimentally. In this report, we present results of measurements of free plasma melatonin and total plasma melatonin in human samples collected between 2000–1000 h and compare the results with saliva melatonin levels in samples collected simultaneously.

	Materials and Methods

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Ten subjects (five men and five women; mean age, 22.2 yr; range 18–30 yr) spent 1 night at the Center for Sleep Research at the Queen Elizabeth Hospital. The subjects were screened for general health using a questionnaire and were excluded if they were smokers, had current health or sleep problems, or were taking medication known to affect melatonin production, e.g. ß-adrenergic blockers. All subjects gave their informed consent and were compensated for the inconvenience associated with their participation. The studies were approved by the Queen Elizabeth Hospital ethics of human experimentation committee using guidelines established by the National Health and Medical Research Council of Australia. Blood was collected just before the collection of saliva samples via an indwelling cannula in an antecubital vein at hourly intervals from 2000–1000 h, and plasma was harvested and stored frozen until assayed. Saliva was collected by chewing for 2 min on a small piece of parafilm. The saliva samples were frozen for at least 24 h, then thawed and centrifuged to clear the samples of mucins and cellular debris. The supernatant was stored frozen until subsequent analysis. The samples used here were collected as part of another larger study on the validation of salivary melatonin measurement as a phase marker for circadian rhythm studies (4).

Saliva melatonin was assayed using a direct RIA of 200 µL saliva using standards and reagents provided by Buhlmann Laboratories (Allschwil, Switzerland). This assay uses the G280 antibody developed in this laboratory (5) and [¹²⁵I]2-iodomelatonin as the radioligand (6). Plasma melatonin was assayed using an extraction RIA according to procedures outlined in the Buhlmann melatonin RIA kit. This assay uses small C₁₈ reverse phase columns to extract the melatonin, the G280 antibody, and [¹²⁵I]2-iodomelatonin as the radioligand. The sensitivity of the assay (using 300 µL plasma) is 7.2 pmol/L.

To obtain the free (unbound) fraction of melatonin in plasma, 1 mL plasma was incubated at 25 C for 2 h and pipetted into Centrifree micropartition tubes (Amicon, Beverly, MA). The tubes were then centrifuged at 2000 x g for 45 min at 25 C in a Sorval centrifuge (Sorval, Milan, Italy). Three hundred microliters of the filtrate were added to the C₁₈ columns from the Buhlmann kits and processed according to the kit instructions. An additional 300 µL plasma that were not centrifuged through the Centrifree tubes were analyzed in the same assay.

To investigate some of the properties of the binding of melatonin to plasma proteins, dilutions of plasma in Tris-HCl buffer (50 mmol/L; pH 7.4) were incubated at 25 C for 2 h with 10,000 cpm [¹²⁵I]2-iodomelatonin in a final volume of 1 mL. Aliquots (900 µL) were then added to Centrifree tubes and centrifuged for 45 min at 25 C. Aliquots of the filtrate (300 µL) were counted in a -counter. To estimate the affinity of binding, 50 µL plasma were incubated with 10,000 cpm [¹²⁵I]2-iodomelatonin and together with unlabeled melatonin (10^-6-10^-3 mol/L) were processed as described above.

	Results

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Melatonin was detected in the filtrate of human plasma samples collected throughout the night. The peak concentrations for the subjects ranged from 45–200 pmol/L (mean SEM, 106 ± 17 pmol/L). Figure 1 shows the mean total melatonin concentrations and the free melatonin levels in the 10 subjects. Unbound melatonin represented approximately 23% of the total plasma concentration. Figure 2 shows the saliva melatonin concentration plotted with the free melatonin concentration. The correlation between the saliva and free melatonin levels was significant (r = 0.84; n = 127; P < 0.05). The free/total plasma ratio and the saliva/total plasma ratios did not vary throughout the night. There was a significant correlation (r = 0.69; P < 0.05) between the free/total plasma ratio and saliva/total plasma ratios from samples taken at 0300 h (i.e. the time of peak secretion).

View larger version (23K): [in this window] [in a new window]   Figure 1. Plasma total, plasma free, and saliva melatonin levels in 10 subjects between 2000–1000 h. The data are the mean ± SEM (picomoles per L). Plasma total melatonin is shown in the top panel (•); plasma free melatonin () and saliva melatonin () are shown in the lower panel.

View larger version (19K): [in this window] [in a new window]   Figure 2. The correlation between plasma free melatonin and saliva melatonin in 10 subjects. The data are shown as picomoles per L.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This report presents the first direct evidence that endogenous melatonin in human plasma is bound to high mol wt serum proteins. The fraction bound to proteins varied between 61–85% in the 10 subjects studied, but did not vary throughout the night. The highly significant correlation between the unbound fraction of melatonin in plasma and the salivary melatonin levels as well as the similar ratios strongly suggest that the saliva melatonin levels are indeed a reflection of the free plasma fraction, as previously hypothesized.

Previously, Cardinali et al. (2) studied plasma melatonin binding in vitro using very low specific activity tritiated melatonin (200 mCi/mmol) and equilibrium dialysis. They found that binding was highest at 4 C (78%) and lowest at 37 C (61%). Addition of melatonin up to 1.5 mmol/L failed to inhibit the binding. Melatonin bound only to albumin and not to -globulin, ß-globulin, -globulin, or fibrinogen. Subsequently, Pardridge and Meitus (7) used higher specific activity tritiated melatonin (29 Ci/mmol), again with equilibrium dialysis at 37 C, and confirmed the original observation of 61% binding to plasma proteins. Albumin-bound melatonin was also observed to cross the blood-brain barrier (7). A recent study by Morin et al. (8) again confirmed melatonin binding to albumin, but provided new evidence for melatonin binding to the acute phase protein, ₁-acid glycoprotein. The equilibrium association constants for albumin and ₁-acid glycoprotein were 1.5 and 27 mmol/L, respectively. This binding affinity is up to 6 orders of magnitude lower than the affinity of other hormones for their specific binding globulins (9). In our studies we used the very high specific activity radioiodinated melatonin analog [¹²⁵I]2-iodomelatonin (2000 Ci/mmol) and observed somewhat higher binding to plasma (84% at 25 C). The higher degree of binding to plasma proteins is similar to that found when the binding of the iodinated analog is compared to that of authentic melatonin at tissue melatonin receptors (10). Addition of unlabeled authentic melatonin, even at concentrations up to 1 mmol/L, decreased binding by only 37%, indicating a very low affinity constant for the binding.

The physiological significance of the plasma binding of melatonin has not been seriously considered previously. In other endocrine systems, the level of plasma binding of hormones profoundly affects the biological activity of the hormones. Whether this is the case for melatonin remains to be determined. An argument against an important physiological role of plasma binding of melatonin is the very low affinity binding measured (on the order of 1 mmol/L), which is many orders of magnitude lower than those of many other hormones. Thus, binding to proteins may have little real effect in buffering transfer of melatonin across membranes or preventing binding to the high affinity melatonin receptors.

The presumptive protein responsible for binding melatonin in blood is albumin, and it is established that the concentration of this protein is lower in elderly subjects than in younger subjects (11, 12). There are reports in the literature suggesting that melatonin secretion is lower in the elderly (13) and that this may be responsible in part for the high incidence of sleep disturbance in this age group. It might be predicted, then, that elderly subjects will have less plasma-bound melatonin than young people. This link becomes a tenuous one, however, if the level of (biologically active?) free melatonin is, in fact, increased in the elderly due to decreased albumin binding. Nevertheless, the recent discoveries of low capacity melatonin binding to ₁-acid glycoprotein (8) and interactions between this binding and albumin binding raise the possibility that a number of physiological conditions leading to alterations in the levels of these proteins may change free melatonin levels in the circulation. Clearly, there is a need for further studies on the free/total melatonin ratio within and across age groups using either the direct approach used here or the saliva/plasma melatonin ratio. Albumin is also a carrier protein for many drugs (14, 15), and other hormones and studies are required to investigate the interactions between melatonin and drugs/hormones, and their possible consequences.

In conclusion, we have measured unbound endogenous melatonin in plasma for the first time and shown a close relationship between circulating free and salivary melatonin levels. The percentage of binding of melatonin to plasma proteins did not vary throughout the night despite large changes in the total concentration.

	Footnotes

 
¹ This work was supported by a grant from the National Health and Medical Research Council of Australia.

Received July 9, 1997.

Revised November 7, 1997.

Accepted November 13, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Vaughan GM, Pelham RW, Pang SF, et al. 1976 Nocturnal elevation of plasma melatonin and urinary 5-hydroxyindoleacetic acid in young men: attempts at modification by brief changes in environmental lighting and sleep and by autonomic drugs. J Clin Endocrinol Metab. 42:752–764.[Abstract]
2. Cardinali DP, Lynch HJ, Wurtman RJ. 1972 Binding of melatonin to human and rat plasma proteins. Endocrinology. 91:1213–1218.[Medline]
3. Vakkuri O. 1985 Diurnal rhythm of melatonin in human saliva. Acta Physiol Scand. 124:409–412.[Medline]
4. Voultsios A, Kennaway DJ, Dawson D. 1997 Salivary melatonin as a circadian phase marker: validation and comparison with plasma melatonin. J Biol Rhythms. 12:457–466.
5. Kennaway DJ, Gilmore TA, Seamark RF. 1982 Effect of melatonin feeding on serum prolactin and gonadotropin levels and the onset of seasonal estrous cyclicity in sheep. Endocrinology. 110:1766–1772.[Medline]
6. Vaughan GM. 1993 New sensitive serum melatonin radioimmunoassay employing the Kennaway G280 antibody: Syrian hamster morning adrenergic response. J Pineal Res. 15:88–103.[Medline]
7. Pardridge WM, Mietus LJ. 1980 Transport of albumin-bound melatonin through the blood-brain barrier. J Neurochem. 34:1761–1763.[CrossRef][Medline]
8. Morin D, Simon N, Depresbrummer P, Levi F, Tillement JP, Urien S. 1997 Melatonin high-affinity binding to alpha-1-acid glycoprotein in human serum. Pharmacology. 54:271–275.[Medline]
9. Mendel CM. 1989 The free hormone hypothesis: a physiologically based mathematical model. Endocr Rev. 10:232–274.[Abstract]
10. Kennaway DJ, Hugel HM, Rowe SA. 1994 Characterization of the chicken brain melatonin-binding protein using iodinated and tritiated ligands. J Pineal Res. 17:137–148.[Medline]
11. Davis D, Grossman SH, Kitchell BB, Shand DG, Routledge PA. 1985 The effects of age and smoking on the plasma protein binding of lignocaine and diazepam. Br J Clin Pharmacol. 19:261–265.[Medline]
12. Viani A, Rizzo G, Carrai M, Pacifici GM. 1992 The effect of ageing on plasma albumin and plasma protein binding of diazepam, salicylic acid and digitoxin in healthy subjects and patients with renal impairment. Br J Clin Pharmacol. 33:299–304.[Medline]
13. Waldhauser F, Weiszenbacher G, Tatzer E, et al. 1988 Alterations in nocturnal serum melatonin levels in humans with growth and aging. J Clin Endocrinol Metab. 66:648–652.[Abstract]
14. Koch Weser J, Sellers EM. 1976 Drug therapy. Binding of drugs to serum albumin (second of two parts). N Engl J Med. 294:526–531.[Medline]
15. Koch Weser J, Sellers EM. 1976 Binding of drugs to serum albumin (first of two parts). N Engl J Med. 294:311–316.[Medline]

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